As a medical diagnosis, a pathological diagnosis is performed. A pathologist diagnoses a disease using a tissue section collected from a human body and informs a clinician of whether or not a therapy and/or a surgery is/are necessary. Based on the patient conditions and pathological diagnosis, a physician determines pharmacotherapeutic strategies and a surgeon determines whether or not a surgery should be performed.
In pathological diagnosis, it is a common practice to prepare a tissue sample by slicing a tissue specimen obtained by evisceration or needle biopsy into a thickness of several micrometers or so and observe the tissue sample at a magnification under a light microscope so as to obtain various findings. In many cases, a specimen is prepared by fixing a collected tissue through dehydration and paraffin blocking, slicing the resultant into a thickness of several micrometers and then removing the paraffin. Here, since the specimen hardly absorbs or scatters any light and is thus nearly colorless and transparent, it is usually stained with a dye prior to being observed.
There have been proposed a variety of staining techniques. In particular, for tissue samples, hematoxylin-eosin staining (HE staining) using two dyes, hematoxylin and eosin, is typically used as staining for observing the morphology of a specimen (Patent Literatures 1 and 2). Hematoxylin stains cell nuclei, calcareous parts, cartilaginous tissues, bacteria and mucus in livid to light blue, while eosin stains cytoplasm, interstitial tissues, various fibers, erythrocytes and keratinocyte in red to dark red. A pathologist makes a diagnosis based on morphological information and staining information, such as changes in the size and shape of cell nuclei and changes in the pattern as a tissue, in a microscope image of a stained tissue sample. Examples of other staining for morphological observation include Papanicolaou staining (Pap staining) used for cytological diagnosis.
Further, in pathological diagnosis, immunological observation in which molecular target staining called immunostaining is performed for confirming the expression of molecular information of a specimen and functional abnormalities such as abnormal expression of a gene or a protein are diagnosed is performed. For immunostaining, for example, a dye staining method using an enzyme (DAB staining) is employed. In DAB staining, an antibody modified with peroxidase, which is capable of allowing DAB (diaminobenzidine) as a substrate to show a color, is used to stain an antigen to be observed with the color and the amount of the antigen is determined by observing the stained antigen. Alternatively, fluorescent labeling may be employed. In fluorescent labeling, the amount of the subject antigen is determined by staining the antigen with an antibody modified with a fluorescent dye and observing the stained antigen.
At present, attempts are being made to simultaneously perform morphological observation and immunological observation of a specimen and, for example, it has been tried to simultaneously perform HE staining for morphological observation and DAB staining for immunological observation (Patent Literature 3). However, since staining with an enzyme label, such as DAB staining, develops a color similar to the color developed by HE staining and the colors developed by HE staining and staining with an enzyme label cannot thus be easily distinguished, there is a problem that such simultaneous observation is difficult. In addition, in DAB staining, since the staining concentration is largely affected by the environmental conditions such as temperature and time, there is a problem that estimation of the actual amount of an antibody or the like from the staining concentration is difficult.
On another front, in pathological diagnosis, fluorescent labeling using a fluorescent label is also performed. This method characteristically has excellent quantitative capability as compared to DAB staining. In cases where pathological diagnosis and morphological observation are simultaneously performed using a fluorescent label, there is a problem that the results are likely to be affected by the fluorescence of the staining agent used for tissue staining. As a countermeasure, for example, a fluorescent dye which has peaks of excitation and emission wavelengths in the infrared region and is thus not likely to be affected by visible light can be used (Patent Literature 4). Alternatively, for example, the excitation and emission wavelengths of a staining agent for morphological observation and those of a fluorescent label for immunostaining can be shifted.
Meanwhile, it is known that dyes are easily deteriorated generally in the short-wavelength side, particularly in the ultraviolet region. Considering the effect on deterioration of a staining agent and a fluorescent label, it is desired that they be excited in the visible region rather than in the ultraviolet region.
As fluorescent labels, dyes, inorganic nanoparticles (that may also be referred to as “semiconductor nanoparticle”, “quantum dot” or the like) and aggregates thereof are known to be utilized (Patent Document 5). Thereamong, inorganic nanoparticles are not suitable as the above-described fluorescent label to be excited in the visible region; therefore, it is difficult to utilize inorganic nanoparticles. On the other hand, fluorescent dyes and aggregates thereof can be utilized as the above-described fluorescent label to be excited in the visible region. It has been reported that, between a fluorescent dye and an aggregate thereof, the light-resistant performance is more improved by the latter (Patent Document 6). Furthermore, between a dye and an aggregate thereof, the brightness of individual dye is higher in the aggregate.